Rapid assay for blood meal identification in Aedes albopictus, the Asian tiger mosquito

Blood meal analysis (BMA) is a useful tool for researchers studying disease vectors such as mosquitoes. Understanding the origin of blood meals informs what species participate in disease transmission cycles as well as the disease risk posed to humans. In current BMA, vertebrate primers amplify multiple copy DNA from a blood meal, commonly mtDNA cytochrome b, which is then sequenced and blasted against known sequences in GenBank to identify its source. This technique, however, is very time consuming and costly as each individual sample must be sequenced and mixed blood meals cloned. Further, when applied to Aedes albopictus standard primers amplified mosquito DNA making direct sequencing impossible. To address these problems we have designed a rapid assay allowing easy identification of human blood meals as well as mixed meals between humans and nonhumans. The assay consists of a nested PCR with an internal multiplex using a blocking primer. In the blocking technique a primer with a 3’ mismatch is used in conjunction with the Stoffel Taq fragment, which lacks 5’-3’ exonuclease activity, to anneal to a specific template and prevent extension. The blocking primer approach was necessary to prevent the amplification of a nuclear cytb pseudogene in humans. To our knowledge this is the first application of the blocking technique in conjunction with a nested PCR to identify blood meals in arthropod vectors. While this assay was designed for use in Aedes albopictus, it may have a broader application in other anthropophilic mosquitoes, particularly in urban environments.